Compositions and methods of treating liver disease

ABSTRACT

Disclosed are methods of treating or reducing the occurrence of a steatohepatitis disorder. The disorder may include, for example, NASH, parenteral nutrition associated liver disease (PNALD), or genetic forms of liver disease. The method may comprise the step of administering a composition comprising obeticholic acid to an individual in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 62/471,371, filed Nov. 4, 2016, and 62/517,414,filed Jun. 9, 2016, the contents of each are incorporated by referencein their entirety for all purposes.

BACKGROUND

PNALD is the most devastating complication of long-term PN that occursin the majority of children receiving PN (Kumar and Teckman, 2015; Orsoet al., 2016; Zambrano et al., 2004). Because its progression istypically insidious and its long-term consequences are generallyunderappreciated, PNALD is often recognized too late, when liver injuryis irreversible. It is histologically characterized by intrahepaticcholestasis but can progress to fibrosis and cirrhotic liver failurewith continued exposure to PN. There are no established ameliorativestrategies for PNALD and PNALD is the leading indication for livertransplantation in infants (El Kasmi et al., 2013).

BRIEF SUMMARY

Disclosed are methods of treating or reducing the occurrence of asteatohepatitis disorder. The disorder may include, for example, NASH,parenteral nutrition associated liver disease (PNALD), or genetic formsof liver disease. The method may comprise the step of administering acomposition comprising obeticholic acid to an individual in needthereof.

BRIEF DESCRIPTION OF THE DRAWING

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Obeticholic acid treatment rescue PNALD like pathology in vitroA. Bright-field image of and survival rate (%) of HLO treated 0 (blackline), 400 μM Intralipid (Int; light blue line), 400 μM Int with OCA(gray line), and 400 μM Int with 40 ng/ml FGF19 (pink line). Arrowheadsindicate the dead organoids with clumped morphology in the images.Survival rate (%) of 0 (black bar), 800 μM OA (blue bar), 800 μMOA+FGF19 (pink bar) at the same time point. The survival of 800 μM OAHLO was significantly improved by FGF19 addition. B. Live-cell imagingof ROS (green) and nuclear (blue). OCA (gray bar) and FGF19 (pink bar)inhibit the ROS production caused by 400 μM Int (light blue bar).C.Intralipid. The stiffness of 0 (gray bar), 800 μM OA (blue bar), 800 μMOA+FGF19 (pink bar) treated HLO was assessed by AFM. The stiffness of800 μM OA HLO was significantly softened by FGF19 addition.

FIG. 2. Lipids accumulation in the organoid in the presence and absenceof 400 μM Intralipid.

FIG. 3. Bright-field image of liver organoids in the culture ofnon-treatment, 800 μM of OA alone, and 800 μM of OA with 40 ng/m1 FGF19.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms and expressions used herein have the ordinary meaning as isaccorded to such terms and expressions with respect to theircorresponding respective areas of inquiry and study except wherespecific meanings have otherwise been set forth herein.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a method” includesa plurality of such methods and reference to “a dose” includes referenceto one or more doses and equivalents thereof known to those skilled inthe art, and so forth.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to an animal that is the object of treatment,observation and/or experiment. Generally, the term refers to a humanpatient, but the methods and compositions may be equally applicable tonon-human subjects such as other mammals. In some embodiments, the termsrefer to humans. In further embodiments, the terms refer to children.

“Therapeutically effective amount” relates to the amount or dose of anactive compound or composition described herein that will lead to one ormore therapeutic effect, in particular, a desired beneficial effect. Atherapeutically effective amount of a substance can vary according tofactors such as the disease state, age, sex, and weight of the subject,and the ability of the substance to elicit a desired response in thesubject. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The term “parenteral nutrition,” or “intravenous feeding,” is a methodof providing nutrition into an individual via intravenousadministration. Depending on which vein is used, this procedure is oftenreferred to as either total parenteral nutrition (TPN) or peripheralparenteral nutrition (PPN).

The phrase “pharmaceutically acceptable,” as used in connection withcompositions of the disclosure, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to asubject (e.g., human) In certain embodiments, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency of aFederal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in mammals (e.g.,humans).

The terms “pharmaceutically acceptable salts” or “a pharmaceuticallyacceptable salt thereof” refer to salts prepared from pharmaceuticallyacceptable, non-toxic acids or bases. Suitable pharmaceuticallyacceptable salts include metallic salts, e.g., salts of aluminum, zinc,alkali metal salts such as lithium, sodium, and potassium salts,alkaline earth metal salts such as calcium and magnesium salts; organicsalts; salts of free acids and bases; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as The Merck Index. Any suitableconstituent can be selected to make a salt of an active drug discussedherein, provided that it is non-toxic and does not substantiallyinterfere with the desired activity. In addition to salts,pharmaceutically acceptable precursors and derivatives of the compoundscan be employed. Pharmaceutically acceptable amides, lower alkyl esters,and protected derivatives of the disclosed actives can also be suitablefor use in the compositions and methods disclosed herein. A salt of acompound of this disclosure may be formed between an acid and a basicgroup of the compound, such as an amino functional group, or a base andan acidic group of the compound, such as a carboxyl functional group.According to another embodiment, the compound is a pharmaceuticallyacceptable acid addition salt. Acids commonly employed to formpharmaceutically acceptable salts include inorganic acids such ashydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid and phosphoric acid, as well as organic acids suchas para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaricacid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconicacid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid,ethanesulfonic acid, benzenes ulfonic acid, lactic acid, oxalic acid,para-bromophenylsulfonic acid, carbonic acid, succinic acid, citricacid, benzoic acid and acetic acid, as well as related inorganic andorganic acids. Such pharmaceutically acceptable salts thus includesulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caprate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate,xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate and other salts. In one embodiment,pharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, andespecially those formed with organic acids such as maleic acid.

The terms “treat,” “treating” or “treatment,” as used herein, refers tomethods of alleviating, abating or ameliorating a disease or conditionsymptoms, preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

The term “carrier” applied to pharmaceutical compositions of thedisclosure refers to a diluent, excipient, or vehicle with which anactive compound is administered. Such pharmaceutical carriers can besterile liquids, such as water, saline solutions, aqueous dextrosesolutions, aqueous glycerol solutions, and oils, including those ofpetroleum, animal, vegetable, or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” (any edition).

The term “compound,” as used herein, is also intended to include anysalts, solvates, or hydrates thereof.

Applicant has discovered novel methods for the treatment of liverdisease in an individual in need thereof, which may includeadministration of a disclosed compound to an individual in need thereof.

In one aspect, a method of treating or reducing the occurrence of asteatohepatitis disorder is disclosed. The disorder may include, forexample, NASH, parenteral nutrition associated liver disease (PNALD), orgenetic forms of liver disease. The method may comprise the step ofadministering a composition comprising obeticholic acid to an individualin need thereof.

In one aspect, the composition may be administered in an amountsufficient to reduce or prevent the occurrence of liver cell fibrosis.

In one aspect, the composition may be administered prior to, following,or during administration of parenteral nutrition to said individual inneed thereof.

In one aspect, a composition is disclosed, wherein the composition maycomprise total parenteral nutrition (TPN) composition or peripheralparenteral nutrition (PPN) composition; and obeticholic acid.

In a further aspect, an article of manufacture is disclosed. The articleof manufacture may comprise a container, a composition as describedherein, the composition being in a dosage form, and instructions foradministering the dosage form to an individual diagnosed or suspected ofhaving or developing a liver disease as disclosed herein.

Dosage

In one aspect, an agent disclosed herein may be present in an amount offrom about 0.5% to about 95%, or from about 1% to about 90%, or fromabout 2% to about 85%, or from about 3% to about 80%, or from about 4%,about 75%, or from about 5% to about 70%, or from about 6%, about 65%,or from about 7% to about 60%, or from about 8% to about 55%, or fromabout 9% to about 50%, or from about 10% to about 40%, by weight of thecomposition.

The compositions may be administered in oral dosage forms such astablets, capsules (each of which includes sustained release or timedrelease formulations), pills, powders, granules, elixirs, tinctures,suspensions, syrups, and emulsions. They may also be administered inintravenous (bolus or infusion), intraperitoneal, subcutaneous,intralesional, or intramuscular forms all utilizing dosage forms wellknown to those of ordinary skill in the pharmaceutical arts. Thecompositions may be administered by intranasal route via topical use ofsuitable intranasal vehicles, or via a transdermal route, for exampleusing conventional transdermal skin patches. A dosage protocol foradministration using a transdermal delivery system may be continuousrather than intermittent throughout the dosage regimen.

In one aspect, the compounds may be administered at the rate of 100 μgto 1000 mg per day per kg of body weight. Orally, the compounds may besuitably administered at the rate of about 100, 150, 200, 250, 300, 350,400, 450, or 500 μg to about 1, 5, 10, 25, 50, 75, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000 mg per day per kg of body weight. Therequired dose can be administered in one or more portions. For oraladministration, suitable forms are, for example, tablets, gel, aerosols,pills, dragees, syrups, suspensions, emulsions, solutions, powders andgranules; one method of administration includes using a suitable formcontaining from 1 mg to about 500 mg of active substance. In one aspect,administration may comprise using a suitable form containing from about1, 2, 5, 10, 25, or 50 mg to about 100, 200, 300, 400, 500 mg of activesubstance.

A dosage regimen will vary depending upon known factors such as thepharmacodynamic characteristics of the agents and their mode and routeof administration; the species, age, sex, health, medical condition, andweight of the patient, the nature and extent of the symptoms, the kindof concurrent treatment, the frequency of treatment, the route ofadministration, the renal and hepatic function of the patient, and thedesired effect. The effective amount of a drug required to prevent,counter, or arrest progression of a symptom or effect of a musclecontracture can be readily determined by an ordinarily skilledphysician.

The pharmaceutical compositions may include suitable dosage forms fororal, parenteral (including subcutaneous, intramuscular, intradermal andintravenous), transdermal, sublingual, bronchial or nasaladministration. Thus, if a solid carrier is used, the preparation may betableted, placed in a hard gelatin capsule in powder or pellet form, orin the form of a troche or lozenge. The solid carrier may containconventional excipients such as binding agents, fillers, tabletinglubricants, disintegrants, wetting agents and the like. The tablet may,if desired, be film coated by conventional techniques. Oral preparationsinclude push-fit capsules made of gelatin, as well as soft, scaledcapsules made of gelatin and a coating, such as glycerol or sorbitol.Push-fit capsules can contain active ingredients mixed with a filler orbinders, such as lactose or starches, lubricants, such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers. If a liquid carrier is employed, the preparation may be inthe form of a syrup, emulsion, soft gelatin capsule, sterile vehicle forinjection, an aqueous or non-aqueous liquid suspension, or may be a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Liquid preparations may contain conventional additives such assuspending agents, emulsifying agents, wetting agents, non-aqueousvehicle (including edible oils), preservatives, as well as flavoringand/or coloring agents. For parenteral administration, a vehiclenormally will comprise sterile water, at least in large part, althoughsaline solutions, glucose solutions and like may be utilized. Injectablesuspensions also may be used, in which case conventional suspendingagents may be employed. Conventional preservatives, buffering agents andthe like also may be added to the parenteral dosage forms. For topicalor nasal administration, penetrants or permeation agents that areappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art. Thepharmaceutical compositions are prepared by conventional techniquesappropriate to the desired preparation containing appropriate amounts ofthe active ingredient, that is, one or more of the disclosed activeagents or a pharmaceutically acceptable salt thereof according to theinvention.

The dosage of an agent disclosed herein used to achieve a therapeuticeffect will depend not only on such factors as the age, weight and sexof the patient and mode of administration, but also on the degree ofinhibition desired and the potency of an agent disclosed herein for theparticular disorder or disease concerned. It is also contemplated thatthe treatment and dosage of an agent disclosed herein may beadministered in unit dosage form and that the unit dosage form would beadjusted accordingly by one skilled in the art to reflect the relativelevel of activity. The decision as to the particular dosage to beemployed (and the number of times to be administered per day) is withinthe discretion of the physician, and may be varied by titration of thedosage to the particular circumstances of this invention to produce thedesired therapeutic effect.

Routes of Administration

Any suitable route of administration can be employed for providing thepatient with an effective dosage of the disclosed compositions. Forexample, oral, rectal, transdermal, parenteral (subcutaneous,intramuscular, intravenous), intrathecal, topical, inhalable, and likeforms of administration can be employed. Suitable dosage forms includetablets, troches, dispersions, suspensions, solutions, capsules,patches, and the like. Administration of medicaments prepared from thecompounds described herein can be by any suitable method capable ofintroducing the compounds into the bloodstream. In some embodiments, theformulations can contain a mixture of active compounds withpharmaceutically acceptable carriers or diluents known to those of skillin the art.

The compositions can be prepared in any desired form, for example,tables, powders, capsules, injectables, suspensions, sachets, cachets,patches, solutions, elixirs, and aerosols. Carriers such as starches,sugars, microcrystalline cellulose, diluents, granulating agents,lubricants, binders, disintegrating agents, and the like can be used inoral solid preparations. In certain embodiments, the compositions areprepared as oral solid preparations (such as powders, capsules, andtablets). In certain embodiments, the compositions are prepared as oralliquid preparations. In some embodiments, the oral solid preparationsare tablets. If desired, tablets can be coated by standard aqueous ornon-aqueous techniques.

In addition to the dosage forms set out above, one or more disclosedcompounds may also be administered by sustained release, delayedrelease, or controlled release compositions and/or delivery devices.

Pharmaceutical compositions suitable for oral administration can beprovided as discrete units such as capsules, cachets, sachets, patches,injectables, tablets, and aerosol sprays, each containing predeterminedamounts of the active ingredients, as powder or granules, or as asolution or a suspension in an aqueous liquid, a non-aqueous liquid, anoil-in-water emulsion, or a water-in-oil liquid emulsion. Suchcompositions can be prepared by any of the conventional methods ofpharmacy, and may include the step of bringing into association theactive ingredients with a carrier which constitutes one or moreingredients. In general, the compositions may be prepared by uniformlyand intimately admixing the active ingredients with liquid carriers,finely divided solid carriers, or both, and then, optionally, shapingthe product into the desired presentation.

For example, a tablet can be prepared by compression or molding,optionally, with one or more additional ingredients. Compressed tabletscan be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with a binder, lubricant, inert diluent, surface active ordispersing agent. Molded tablets can be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent.

A composition or formulation may be administered to a subjectcontinuously or periodically.

The compositions or fractions thereof typically comprise suitablepharmaceutical diluents, excipients, vehicles, or carriers selectedbased on the intended form of administration, and consistent withconventional pharmaceutical practices. The carriers, vehicles etc. maybe adapted to provide an additive, synergistically effective ortherapeutically effective amount of the active compounds. Suitablepharmaceutical diluents, excipients, vehicles, and carriers aredescribed in the standard text, Remington: The Science and Practice ofPharmacy, and in The United States Pharmacopeia: The National Formulary(USP 24 NF 19) published in 1999. By way of example, for oraladministration in the form of a capsule or tablet, the active componentscan be combined with an oral, non-toxic pharmaceutically acceptableinert carrier such as lactose, starch, sucrose, methyl cellulose,magnesium stearate, glucose, calcium, sulfate, dicalcium phosphate,mannitol, sorbital, and the like. For oral administration in a liquidform, the agents may be combined with any oral, non-toxic,pharmaceutically acceptable inert carrier such as ethanol, glycerol,water, and the like. Suitable binders (e.g. gelatin, starch, cornsweeteners, natural sugars including glucose; natural and syntheticgums, and waxes), lubricants (e.g. sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, and sodiumchloride), disintegrating agents (e.g. starch, methyl cellulose, agar,bentonite, and xanthan gum), flavoring agents, and coloring agents mayalso be combined in the compositions or components thereof.

In one aspect, a pharmaceutical composition may have pH from about 7 to10.

Formulations for parenteral administration of a composition may includeaqueous solutions, syrups, aqueous or oil suspensions and emulsions withedible oil such as cottonseed oil, coconut oil or peanut oil. Dispersingor suspending agents that can be used for aqueous suspensions includesynthetic or natural gums, such as tragacanth, alginate, acacia,dextran, sodium carboxymethylcellulose, gelatin, methylcellulose, andpolyvinylpyrrolidone.

Compositions for parenteral administration may include sterile aqueousor non-aqueous solvents, such as water, isotonic saline, isotonicglucose solution, buffer solution, or other solvents conveniently usedfor parenteral administration of therapeutically active agents. Acomposition intended for parenteral administration may also includeconventional additives such as stabilizers, buffers, or preservatives.

In an embodiment, a solid form pharmaceutical composition is provided(e.g. tablets, capsules, powdered, or pulverized form) comprising one ormore disclosed compounds or salt thereof.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of a composition, such labeling wouldinclude amount, frequency, and method of administration.

Kits

Kits are also provided. In one aspect, a kit may comprise or consistessentially of agents or compositions described herein. The kit may be apackage that houses a container which may contain a compositioncomprising an oxime or pharmaceutically acceptable salt thereof asdisclosed herein, and also houses instructions for administering theagent or composition to a subject. In one aspect, a pharmaceutical packor kit is provided comprising one or more containers filled with one ormore composition as disclosed herein. Associated with such container(s)can be various written materials such as instructions for use, or anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use, orsale for human administration.

As there may be advantages to mixing a component of a compositiondescribed herein and a pharmaceutically acceptable carrier, excipient orvehicle near the time of use, kits in which components of thecompositions are packaged separately are disclosed. For example, the kitcan contain an active ingredient in a powdered or other dry form in, forexample, a sterile vial or ampule and, in a separate container withinthe kit, a carrier, excipient, or vehicle, or a component of a carrier,excipient, or vehicle (in liquid or dry form). In one aspect, the kitcan contain a component in a dry form, typically as a powder, often in alyophilized form in, for example, a sterile vial or ampule and, in aseparate container within the kit, a carrier, excipient, or vehicle, ora component of a carrier, excipient, or vehicle. Alternatively, the kitmay contain a component in the form of a concentrated solution that isdiluted prior to administration. Any of the components described herein,any of the carriers, excipients or vehicles described herein, and anycombination of components and carriers, excipients or vehicles can beincluded in a kit.

Optionally, a kit may also contain instructions for preparation or use(e.g., written instructions printed on the outer container or on aleaflet placed therein) and one or more devices to aid the preparationof the solution and/or its administration to a patient (e.g., one or aplurality of syringes, needles, filters, tape, tubing, alcohol swabs, orthe like). Compositions which are more concentrated than thoseadministered to a subject can be prepared. Accordingly, suchcompositions can be included in the kits with, optionally, suitablematerials (e.g., water, saline, or other physiologically acceptablesolutions) for dilution. Instructions included with the kit can include,where appropriate, instructions for dilution.

In other embodiments, the kits can include pre-mixed compositions andinstructions for solubilizing any precipitate that may have formedduring shipping or storage. Kits containing solutions of one or more ofthe aforementioned active agents, or pharmaceutically acceptable saltsthereof, and one or more carriers, excipients or vehicles may alsocontain any of the materials mentioned above (e.g., any device to aid inpreparing the composition for administration or in the administrationper se). The instructions in these kits may describe suitableindications (e.g., a description of patients amenable to treatment) andinstructions for administering the solution to a patient.

EXAMPLES

Repurposing OCA for PNALD Therapy

In this study, Applicant found that OCA and FGF19 inhibited ROSproduction caused by Intralipid exposure in HLO, leading to animprovement in survival rate of HLO. Moreover, the stiffness of OAtreated HLO, measured by AFM, was decreased by FGF19 exposure. Theseresults provide direct experimental evidence that OCA and FGF19 play apreventive role in the pathogenesis of PNALD organoid model and that thesuppression of ROS production and fibrosis by FXR agonism is the likelymechanism of protection against PNALD. Our study thus highlighted thepotential repurposing of OCA for the treatment of patients with PNALD.

Obeticholic Acid Treatment Improved PNALD-Like Pathology of HLO

PNALD is an iatrogenic fatty liver disease which occurs frequently ininfants provided with parental nutrition (PN) solutions employing alipid emulsion (Intralipid) that provide life-sustaining calories in thesetting of inadequate absorption of enteral nutrients (Kumar andTeckman, 2015; Orso et al., 2016). PNALD has a histological evidence ofsteatosis, cell death (apoptosis), and fibrosis (Nandivada et al.,2013). To recapitulate PNALD pathology in HLO, we reconstitutedcomponents of Intralipid and placed it directly into HLO culture.Similar to OA treatment, significant lipid accumulation was confirmed in400 μM Intralipid treated HLO reflecting a steatosis-like condition(FIG. 2). In parallel, we established an organoid survival assessmentalgorithm by morphological analysis backed by viability assessment(FIGS. 3 and 2, panel A.) and demonstrated that HLO survival of 400 μMIntralipid treated group was severely compromised by 37% after 12 daysof culture (FIG. 2, panel A).

Because there are no established ameliorative strategies, and PNALD isthe leading indication for liver transplantation in infants (El Kasmi etal., 2013), we further investigated preventive and therapeutic effectsof FXR agonist obeticholic acid (OCA) and FGF19 hormone that areactivated by OCA, which have shown great potential in nonalcoholicsteatohepatitis treatment (Dash et al., 2017; Kumar and Teckman, 2015;Neuschwander-Tetri et al., 2015). To implicate the therapeuticsignificance of OCA and FGF19 for PNALD like pathology in HLO, OCA andFGF19 were added to 400 μM Intralipid exposed HLO. By addition of OCAand FGF19 into PNALD-HLO, the number of surviving organoids remarkablyimproved around 83 and 78% in total, respectively. Mechanistically, theaccumulation of reactive oxygen species (ROS) is strongly associatedwith the fatty liver related hepatocyte death (Nandivada et al., 2013;Tsedensodnom and Sadler, 2013). We thus wondered whether OCA and FGF19would inhibit the production of ROS so we sought to confirm that theorywith live-cell imaging with CellROX dye. Consistent with HLO survivalimprovements, ROS production was 60% in 400 μM Intralipid treated HLObut reduced to 20 and 30% by OCA and FGF19 treatments, respectively(FIG. 7, B), suggesting that OCA and FGF19 might have therapeuticpotential against PNALD through suppressing ROS production.

To further determine whether OCA and FGF19 have an impact on fibrosis,HLO stiffness was assessed. Organoid damage induced by Intralipid isextremely significant and treated HLOs for 5 days are not able to bestabilized on a dish, which is a minimal requirement for assessing therigidity by AFM. To circumvent this limitation, the stiffness wasassessed at Dya3. These observations indicate that OCA and FGF19 reducethe progression of fibrosis, potentially alleviating PNALD phenotype inan organoid.

Methods

hPSCs Maintenance. The TkDA3 human iPSC clone used in this study waskindly provided by K. Eto and H. Nakauchi. Human iPSC lines weremaintained as described previously (Takebe et al., 2015; Takebe et al.,2014). Undifferentiated hiPSCs were maintained on feeder-free conditionsin mTeSR1 medium (StemCell technologies, Vancouver, Canada) on platescoated with Matrigel (Corning Inc., NY, USA) at 1/30 dilution at 37° C.in 5% CO₂ with 95% air.

Definitive endoderm induction. Human iPSCs into definitive endoderm wasdifferentiated using previously described methods with slightmodifications (Spence et al., 2011). In brief, colonies of human iPSCswere isolated in Accutase (Thermo Fisher Scientific Inc., MA, USA) and150,000 cells/mL, were plated on Matrigel coated tissue culture plate(VWR Scientific Products, West Chester, Pa.). Medium was changed to RPMI1640 medium (Life Technologies) containing 100 ng/mL Activin A (R&DSystems, MN, USA) and 50 ng/mL bone morphogenetic protein 4 (BMP4; R&DSystems) at day 1, 100 ng/mL Activin A and 0.2% fetal calf serum (FCS;Thermo Fisher Scientific Inc.) at day 2 and 100 ng/mL Activin A and 2%FCS at day 3. Day 4-6 cells were cultured in Advanced DMEM/F12 (ThermoFisher Scientific Inc.) with B27 (Life Technologies) and N2 (Gibco, CA,USA) containing 500 ng/ml fibroblast growth factor (FGF4; R&D Systems)and 3 uM CHIR99021 (Stemgent, MA, USA). Cells were maintained at 37° C.in 5% CO₂ with 95% air and the medium was replaced every day. Spheroidsappeared on the plate at day 7 of differentiation.

HLO induction. At day 7, spheroids and attached cells are gentlypipetted to be delaminated from dishes. They were centrifuged at 800 rpmfor 3 minutes, embedded in a 100% Matrigel drop on the dishes inAdvanced DMEM/F12 with B27, N2 and 2 uM retinoic acid (RA; Sigma, MO,USA) after removing supernatant, and cultured for 4 days. After RAtreatment, spheroids embedded in the Matrigel drop were cultured inHepatocytes Culture Medium (HCM; Lonza, MD, USA) with 10 ng/mLhepatocyte growth factor (HGF; PeproTech, NJ, USA), 0.1 uM Dexamethasone(Dex; Sigma) and 20 ng/mL Oncostatin M (OSM; R&D Systems). Cultures forHLO induction were maintained at 37° C. in 5% CO₂ with 95% air and themedium was replaced every 2-3 days. To analyze HLO (day 20-30),organoids were isolated from Matrigel by scratching and pipetting.

Albumin, IL-6, and P3NP ELISA. To measure albumin secretion level ofHLO, 200 μL of culture supernatant was collected from HLO embedded inMatrigel. For IL-6 and P3NP, 20-30 organoids were seeded and cultured onan ultra-low attachment multiwell plates 96 well plate (Corning). Todefine the exact number of organoids in each well and lastly normalizethe secreted level for IL-6 and P3NP by the number, the organoids werecaptured on The KEYENCE BZ-X710 Fluorescence Microscope. The culturesupernatants were collected at 24 hrs (for albumin), 96 hrs (for IL-6)and 120 hrs (P3NP) time points after the culture and stored at −80° C.until use. The supernatant was centrifuged at 1,500 rpm for 3 min and topellet debris, and the resulting supernatant was assayed with HumanAlbumin ELISA Quantitation Set (Bethyl Laboratories, Inc., TX, USA),Human IL-6 ELISA Kit (Biolegend, CA, USA), and Human N-terminalprocollagen III propeptide, PIIINP ELISA Kit (My BioSource, CA, USA)according to the manufacturer's instructions. Significance testing wasconducted by Student's t-test.

Bile transport activity. Fluorescein diacetate was used for evaluatingbile transport activity in organoids. 10 mg/mL fluorescein diacetate(Sigma) was added into HCM media cultured with HLO and allowed to sitfor 5 minutes and captured using fluorescent microscopy BZ-X710(Keyence, Osaka, Japan).

Phagocyte, lipids, ROS live-cell imaging. After being cultured in anultra-low attachment 6 multi-well plate, 5-10 HLO were picked up andseeded in a Microslide 8 Well Glass Bottom plate (Ibidi, WI, USA) andsubjected to live-cell staining. The following antibodies were used:pHrodo® Red S. aureus Bioparticles® Conjugate for Phagocyte activity(Thermo Fisher Scientific Inc.), BODIPY® 493/503 for lipids (ThermoFisher Scientific Inc.), Di-8-ANEPPS for membrane (Thermo FisherScientific Inc.), and CellROX green reagent for ROS detection (ThermoFisher Scientific Inc.). Nuclear staining was marked by NucBlue LiveReadyProbes Reagent (Thermo Fisher Scientific Inc.). HLO was visualizedand scanned on a Nikon A1 Inverted Confocal Microscope (Japan) using 60×water immersion objectives. The final lipid droplet volume wascalculated by IMARIS8 and normalized by each organoid size. Significancetesting for lipid droplet volume and ROS production (%) was conducted byStudent's t-test.

HE staining and immunohistochemistry. HLO were isolated from Matrigeland fixed in 4% paraformaldehyde and embedded in paraffin. Sections weresubjected to HE and immunohistochemical staining. The following primaryantibodies were used: anti-alpha smooth muscle actin antibody (1:200dilution; abcam, Cambridge, UK), Desmin antibody (Pre-diluted; Roche,Basel, Switzerland), and CD68 antibody (1:200 dilution; abcam).

Flow cytometry. HLO were isolated from 10 Matrigel droplets and washedby 1× PBS. HLO were dissociated to single cells by the treatment ofTrypsin-EDTA (0.05%), phenol red (Gibco) for 10 min After PBS wash, thesingle cells were subjected to flow cytometry with BV421-conjugatedEpcam antibody (BioLegend), PE-conjugated CD166 antibody (eBioscience,Calif., USA), and PE/Cy7-CD68 (eBioscience). DNA was measured bypropidium iodide staining.

LPS and FFA exposure and OCA and FGF19 treatment. 20-30 hLOHLO, whichhad been isolated from Matrigel and washed by 1× PBS, were divided intoeach condition and cultured on an ultra-low attachment 6 multi-wellplates (Corning). HLO were cultured with LPS (Sigma), OA (Sigma), LA(Sigma), SA (Sigma), or PA (Sigma) and collected at day 1 and 3 (for LPSHLO) and at day 3 and 5 (for OA) after the culture. To test theinhibitory effect of OCA (INT-747, MedChem Express, NJ, USA) and humanFGF19 recombinant (Sigma) on HLO, 20-30 HLO were cultured in HCM mediain the presence or absence of oleic acid (800 μM), and 1 μM OCA and 40ng/ml FGF19 were added into 800 μM OA condition. HLO were collected atday 3 for lipids live-cell imaging and at day 5 for stiffnessmeasurement.

Whole mount immunofluorescence. HLO were fixed for 30 min in 4%paraformaldehyde and permeabilized for 15 min with 0.5% Nonidet P-40.HLO were washed by 1× PBS three times and incubated with blocking bufferfor 1 h at room temperature. HLO were then incubated with primaryantibody; anti-alpha smooth muscle actin antibody (1:50 dilution; abcam)overnight at 4° C. HLO were washed by 1× PBS and incubated in secondaryantibody in blocking buffer for 30 min at room temperature. HLO werewashed and mounted using Fluoroshield mounting medium with DAPI (abcam).The stained HLO were visualized and scanned on a Nikon A1 InvertedConfocal Microscope (Japan) using 60× water immersion objectives.

RNA isolation, RT-qPCR. RNA was isolated using the RNeasy mini kit(Qiagen, Hilden, Germany) Reverse transcription was carried out usingthe SuperScriptlll First-Strand Synthesis System for RT-PCR (Invitrogen,CA, USA) according to manufacturer's protocol. qPCR was carried outusing TaqMan gene expression master mix (Applied Biosystems) on aQuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific Inc.). Allprimers and probe information for each target gene was obtained from theUniversal ProbeLibrary Assay Design Center(https://qpcr.probefinder.com/organism.jsp). Significance testing wasconducted by Student's t-test.

HLO stiffness measurement by AFM. HLOs treated with 0, 50200, 1400, 2800ng/mLμM LPSOA were used for stiffness measurement with a AFM (NanoWizardIV, JPK Instruments, Germany). The AFM head with a silicon nitridecantilever (CSC37, k=0.3 N/m, MikroMasch, Bulgaria) was mounted on afluorescence stereo microscope (M205 FA, Leica, Germany) coupled with aZ-axis piezo stage (JPK CellHesion module, JPK Instruments, Germany),which allows the indentation measurement up to the depth of ˜100 μm. Asa substrate for organoids, a fibronectin-coated dish was used. Thetissue culture dish (φ=34 mm, TPP Techno Plastic Products, Switzerland)was first incubated with a 1 μg/mL fibronectin solution (Sigma)) at 4°C. for overnight. Then, the tissue culture dish was washed twice bydistilled water and dried for 1 hour. Thereafter, HLOs incubated withOALPS for 51 days were deposited to the fibronectin-coated dish andincubated for 1 hour at 37° C. The sample dish was then placed onto theAFM stage, and force-distance curves in a 14×14 matrix in a 25×25 μmsquare were measured from each HLO. Finally, Young's moduli (E, Pa) ofHLOs were determined by fitting the obtained force-distance curves withthe modified Hertz model (Sneddon, 1965). Dunn-Holland-Wolfe test wasperformed for significance testings.

THP-1 cell migration assay. THP-1 cell, which was gifted from T. Suzuki,was maintained in Advanced DMEM/F12 (Thermo Fisher Scientific Inc.)containing 10% FBS. THP-1 floating cells were collected, and 200,000cells were added with serum-free Advanced DMEM/F12 to the membranechamber of the CytoSelect™96-Well Cell Migration Assay (5 μm,Fluorometric Format; Cell Biolabs, CA, USA). 10-20 HLO were cultured inHCM media including 0, 400, 800 uM OA with an ultra-low attachment 96multi-well plate (Corning) for three days. To define the exact number oforganoids in each well and lastly normalize the final migrated cells bythe number, the organoids were captured on The KEYENCE BZ-X710Fluorescence Microscope. 150 μL of culture supernatant of HLO wascollected and added to the feeder tray of the kit. The kit was incubatedat 37° C. for 24 h in a 5% CO2 cell culture incubator. Cells that hadmigrated were counted using Countess II FL Automated Cell Counter(Thermo Fisher Scientific Inc.). Significance testing was conducted byStudent's t-test.

Triglyceride assay. For quantitative determination of triglycerides, HLOwere isolated from one Matrigel drop and divided into HCM media in thepresence or absence of oleic acid (800 μM). They were cultured on anultra-Low attachment 6 multi-well plate for three days. Quantitativeestimation of hepatic triglyceride accumulation was performed by anenzymatic assay of triglyceride mass using the EnzyChrom Triglycerideassay kit (Bioassay Systems, CA, USA).

HLO survival assay. HLO were collected from Matrigel and washed by 1×PBS. 30-40 organoids were cultured on an ultra-low attachment 6multi-well plate (Corning). HLO were captured on The KEYENCE BZ-X710Fluorescence Microscope every day. The surviving and dead organoids werecounted manually from the photo. HLO with a rounded configuration wascounted as the surviving while the organoids with out of shape iscounted as dead. To assess the survival rate of OA treated HLO at thesame time point, 3D cell titer glo assay was used (Promega, Wi, USA).

Statistics and reproducibility. Statistical analysis was performed usingunpaired two-tailed Student's t-test, Dunn-Holland-Wolfe test, orWelch's t-test. Results were shown mean±s.e.m.; P values<0.05 wereconsidered statistically significant. N-value refers to biologicallyindependent replicates, unless noted otherwise.

All percentages and ratios are calculated by weight unless otherwiseindicated.

All percentages and ratios are calculated based on the total compositionunless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “20 mm” is intended to mean“about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A method of treating or reducing the occurrence of a steatohepatitisdisorder, including NASH, parenteral nutrition associated liver disease(PNALD), and a genetic form of liver disease, comprising the step ofadministering a composition comprising obeticholic acid to an individualin need thereof.
 2. The method of claim 1, wherein said composition isadministered in an amount sufficient to reduce or prevent the occurrenceof liver cell fibrosis.
 3. The method of claim 1 wherein saidcomposition is administered prior to, following, or duringadministration of parenteral nutrition to said individual in needthereof.
 4. The method of claim 1 wherein said composition comprises abuffer.
 5. The method of claim 1 wherein said composition a carrier. 6.The method of claim 1 wherein said composition is administeredparenterally or intravenously.
 7. A composition comprising a. totalparenteral nutrition (TPN) or peripheral parenteral nutrition (PPN); andb. obeticholic acid.
 8. A kit comprising a container, the composition ofclaim 7 in a dosage form disposed in the container, and instructions foradministering said dosage form to an individual diagnosed or suspectedof having or developing PNALD.